EP3155117A2 - Compositions d'a1-antitrypsine et méthodes de traitement de maladies auto-immunes - Google Patents

Compositions d'a1-antitrypsine et méthodes de traitement de maladies auto-immunes

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Publication number
EP3155117A2
EP3155117A2 EP15805947.7A EP15805947A EP3155117A2 EP 3155117 A2 EP3155117 A2 EP 3155117A2 EP 15805947 A EP15805947 A EP 15805947A EP 3155117 A2 EP3155117 A2 EP 3155117A2
Authority
EP
European Patent Office
Prior art keywords
aat
nucleic acid
seq
region
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15805947.7A
Other languages
German (de)
English (en)
Other versions
EP3155117A4 (fr
Inventor
Terry B. Strom
Maria Koulmanda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beth Israel Deaconess Medical Center Inc
Original Assignee
Beth Israel Deaconess Medical Center Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beth Israel Deaconess Medical Center Inc filed Critical Beth Israel Deaconess Medical Center Inc
Publication of EP3155117A2 publication Critical patent/EP3155117A2/fr
Publication of EP3155117A4 publication Critical patent/EP3155117A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates generally to the treatment of autoimmune disease, e.g., diabetes, e.g., Type 1 and Type 2 diabetes.
  • the treatment methods are varied and can include administration of an a 1 -antitrypsin polypeptide (AAT) conjugated to an Fc region of an immunoglobulin.
  • AAT a 1 -antitrypsin polypeptide
  • Type 1 diabetes is also known as insulin-dependent diabetes mellitus (IDDM) and results from insufficient insulin production.
  • IDDM insulin-dependent diabetes mellitus
  • Type 2 diabetes is known as noninsulin-dependent diabetes mellitus (NIDDM) and is the most common form of the disease, accounting for about 90% of all cases of diabetes.
  • NIDDM noninsulin-dependent diabetes mellitus
  • Type 2 diabetes in the initial phases, is characterized by a suboptimal response to insulin. Although insulin is produced, the ability of a given amount of insulin needed to effect a given decrease in blood glucose is increased.
  • subjects manifest a blunted blood glucose response to insulin, i.e., a state of insulin resistance.
  • the causes of diabetes are not completely understood, although both genetic and environmental factors, such as obesity and lack of exercise, increase the risk.
  • Type 1 diabetes is treated with insulin, although other treatments have been proposed and transplantation of the pancreas or insulin-producing islet cells from the pancreas has been used.
  • Non-pharmaceutical intervention is usually prescribed initially for Type 2 diabetes, e.g., diet modification, weight loss, and exercise. If this is not successful, subjects are then generally treated with one of three different types of drugs: drugs that stimulate the release of insulin from the pancreas; drugs that increase a subject's sensitivity to insulin; and drugs that directly affect the circulating levels of glucose (e.g., drugs that decrease the production of glucose from the liver or increase its uptake by muscles).
  • a subject may be prescribed a sulfonylurea, an a-glucosidase inhibitor, metformin (GLUCOPHAGE TM ), or troglitazone (REZULIN TM ).
  • GLUCOPHAGE TM metformin
  • REZULIN TM troglitazone
  • insulin is also used.
  • some subjects manifest exhaustion of the insulin producing apparatus and thereby require insulin therapy.
  • bariatric surgical procedures have been used with some success in those with extreme obesity and Type 2 diabetes.
  • none of the current treatment strategies are optimal, and there is a great need for better ways to treat subjects who have diabetes or who are at risk of developing diabetes.
  • the present invention is based, in part, on the discovery that a methionine-rich protein, e.g., AAT, can be purified without oxidizing its methionine residues. Further, the circulating half-life of AAT can be increased by conjugating it to an Fc region of an immunoglobulin and can be used in compositions and methods to treat autoimmune disease, e.g., diabetes, e.g., Type 1 and Type 2 diabetes.
  • autoimmune disease e.g., diabetes, e.g., Type 1 and Type 2 diabetes.
  • the present disclosure provides purified chimeric AAT-Fc polypeptides, e.g., recombinant polypeptides, comprising AAT conjugated to an Fc region of an immunoglobulin that are surprisingly more active, i.e., more strongly inhibit serine proteases, than wild type or commercial preparations of AAT. Accordingly, in one aspect, the present specification provides chimeric AAT-Fc polypeptides, e.g., recombinant polypeptides, comprising AAT conjugated to an Fc region of an immunoglobulin.
  • AAT can comprise an amino acid sequence that is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2.
  • the Fc region of the amino acid sequence is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2.
  • immunoglobulin can comprise an amino acid sequence that is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or 6.
  • AAT conjugated to an Fc region of an immunoglobulin can comprise an amino acid sequence that is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 10, 12, 14, 16, or 18.
  • compositions comprising a polypeptide comprising AAT conjugated to an Fc region of an immunoglobulin are provided with a pharmaceutically acceptable carrier.
  • nucleic acid molecules encoding a polypeptide comprising AAT conjugated to an Fc region of an immunoglobulin are described.
  • AAT can include an amino acid sequence that is at least 90%>, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2.
  • the nucleic acid sequence encoding AAT is at least 90%>, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: l .
  • the Fc region of an immunoglobulin can comprise an amino acid sequence that is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or 6.
  • the nucleic acid sequence encoding the Fc region of the immunoglobulin is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3 or 5.
  • AAT conjugated to an Fc region of an immunoglobulin comprises an amino acid sequence that is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8, 10, 12, 14, 16, or 18, or a nucleic acid sequence that is at least 90%, e.g., at least 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7, 9, 11, 13, 15, or 17.
  • vectors comprising a nucleic acid molecule encoding a polypeptide comprising AAT conjugated to an Fc region of an immunoglobulin.
  • Pharmaceutical compositions comprising the vector are also provided.
  • the vector can also be comprised within a cell, e.g., bacterial cell, insect cell, mammalian, e.g., human, cell.
  • methods of purifying and isolating methionine -rich proteins e.g., AAT conjugated or not conjugated to an Fc region of an immunoglobulin or linked with other protein(s) are described.
  • the methods can include providing a cell comprising a nucleic acid molecule encoding a polypeptide comprising AAT or AAT conjugated to an Fc region of an immunoglobulin or linked with other protein(s); and culturing the cell under conditions sufficient to produce AAT or AAT conjugated to an Fc region of an immunoglobulin or linked with other protein(s), e.g., in growth media with at least 10 mM, e.g., at least 20 mM, 50 mM, 100 mM, 200 mM, or 500 mM, methionine, thereby producing AAT or AAT conjugated to an Fc region of an
  • the methods include purifying or isolating AAT or AAT conjugated to an Fc region of an immunoglobulin or linked with other protein(s) from the cell by including at least 10 mM, e.g., at least 20 mM, 50 mM, 100 mM, 200 mM, or 500 mM, methionine in standard purification reagents and/or isolation reagents, e.g., extraction buffers, wash buffers, and/or elution buffers, supplemented with methionine.
  • standard purification reagents and/or isolation reagents e.g., extraction buffers, wash buffers, and/or elution buffers, supplemented with methionine.
  • autoimmune disease e.g., diabetes, e.g., Type 1 and Type 2 diabetes
  • the methods comprise administering, e.g., subcutaneously, intraperitoneally, intramuscularly, orally, or by infusion, to a subject a therapeutically effective amount of AAT that is conjugated to an Fc region of an immunoglobulin, to thereby treat autoimmune disease, e.g., diabetes, e.g., Type 1 and Type 2 diabetes, in the subject.
  • the methods can further include treating the subject with insulin.
  • AAT conjugated to an Fc region of an immunoglobulin, as described herein to manufacture a medicament to treat autoimmune disease, e.g., diabetes, e.g., Type 1 and Type 2 diabetes.
  • the methods further comprise selecting the subject, wherein selecting the subject comprises providing a sample, e.g., serum, plasma, or blood, from the subject; assaying the sample to determine a level of glucose in the sample to obtain a test value; comparing the test value to a reference value; and selecting the subject (e.g., selecting the subject for treatment) if the test value is greater than or in certain instances about the same as the reference value.
  • selecting the subject comprises providing a sample, e.g., serum, plasma, or blood, from the subject; assaying the sample to determine a level of glucose in the sample to obtain a test value; comparing the test value to a reference value; and selecting the subject (e.g., selecting the subject for treatment) if the test value is greater than or in certain instances about the same as the reference value.
  • treatment or “treating” is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following:
  • treatment is a reduction of pathological consequence of autoimmune disease.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • FIG. 1 is a schematic diagram showing human AAT fused to a Fc region of a human IgG.
  • FIG. 2 is a schematic diagram showing three different AAT-Fc constructs, with FIG. 2A representing Construct 1 (SEQ ID NO: 8) and Construct 4 (SEQ ID NO: 14), FIG. 2B representing Construct 2 (SEQ ID NO: 10) and Construct 5 (SEQ ID NO: 16), and FIG. 2C representing Construct 3 (SEQ ID NO: 12 and Construct 6 (SEQ ID NO: 18).
  • FIG. 3 is a map of a pFUSE expression vector used to express AAT conjugated to a Fc region of an IgG.
  • FIG. 4 is a schematic diagram of restriction sites in Constructs 1 to 6 used to integrate them into pFUSE expression vectors.
  • FIG. 5 is a panel of two line graphs showing the stability of AAT in acidic conditions (pH 2.1).
  • FIG. 6 is a line graph comparing elastase inhibitory activity of AAT-Fc
  • FIG. 7 is a panel of two line graphs comparing elastase inhibitory activity of AAT-Fc Construct 1 (SEQ ID NO:8) with AAT-Fc Construct 3 (SEQ ID NO: 12).
  • AAT is a serpin that acts as an inhibitor of various serine proteases, but its primary target is elastase. In the absence of AAT, elastase is free to break down elastin, which leads to loss of elasticity of the lungs and results in respiratory complications such as emphysema and chronic obstructive pulmonary disease (COPD).
  • AAT irreversibly inhibits trypsin, chymotrypsin, and plasminogen activator.
  • An aberrant form inhibits insulin-induced nitric oxide synthesis in platelets, decreases coagulation time, and has proteolytic activity against insulin and plasmin (Tanaka et al., Jpn J Cancer Res 82:693- 700, 1991; and Niemann et al, Matrix 12:233-41, 1992).
  • AAT restores normoglycemia in new onset diabetic non-obese diabetic (NOD) mice, a daunting and clinically predictive model for Type 1 diabetes (Koulmanda et al., Proc Natl Acad Sci USA 105: 16242-7, 2008). Many agents have proven effective in preventing frank diabetes when given after early signs of autoimmunity are present, but very few of these agents are effective after the onset of hyperglycemia. Some, but not many, of these agents work after establishment of significant islet cell damage in advance of elevated blood glucose levels.
  • AAT is commercially available from Baxter International Inc., which markets AAT as ARALAST TM for the treatment of chronic augmentation therapy in patients with hereditary emphysema.
  • ARALAST TM is prepared from large pools of human plasma by using the Cohn-Oncley cold alcohol fractionation process, followed by purification steps including polyethylene glycol and zinc chloride precipitation and ion exchange chromatography.
  • ARALAST TM contains AAT with a truncated C-terminal lysine, whereby the Lys394 residue has been removed. Because the metabolic half- life of ARALAST TM is only about 5.9 days, dosing is required approximately once weekly ⁇ e.g., with a dosage of 60 mg/kg body weight).
  • ARALAST TM may also contain trace amounts of IgA. Patients with known antibodies against IgA, which can be present in patients with selective or severe IgA deficiency, have a greater risk of developing potentially severe hypersensitivity and anaphylactic reactions. ARALAST TM is contraindicated in patients with antibodies against IgA due to risk of severe hypersensitivity.
  • the present disclosure provides purified, chimeric, recombinant AAT
  • polypeptides that are more active and have longer in vivo circulating half-lives than commercial AAT.
  • heterologous polypeptides of AAT conjugated to an Fc region of an immunoglobulin ⁇ e.g. , a subclass of antibodies that lacks the heavy chain variable region) are described.
  • AAT can be conjugated to an Fc region of an immunoglobulin ⁇ e.g. , a subclass of antibodies that lacks the heavy chain variable region
  • immunoglobulin molecule of any class ⁇ e.g., IgG, IgM, IgA, IgD, and IgE).
  • a chimeric, recombinant fusion protein consisting of two AATs conjugated to an Fc region of a human IgGl molecule and methods of making and using such proteins.
  • Skilled practitioners will appreciate that the chimeric AAT-Fc polypeptides and methods may be modified to involve a variant or an active fragment of AAT and an Fc region of an immunoglobulin molecule of isotype IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or IgM.
  • AAT can include a full-length, soluble form of the enzyme, or a portion or other mutant thereof that retains sufficient activity to reduce activity of the target of interest to a clinically useful extent, e.g., elastase.
  • AAT can be conjugated to an Fc region of an immunoglobulin molecule at the N-terminal or C-terminal end of AAT.
  • the Fc region may include a mutation that inhibits complement fixation and Fc receptor binding, or it may be lytic (i.e., able to bind complement or to lyse cells via another mechanism, such as antibody-dependent complement lysis (ADCC)).
  • ADCC antibody-dependent complement lysis
  • AAT can be conjugated or fused to an Fc region of an
  • immunoglobulin e.g., IgGl .
  • AAT can be conjugated directly to an Fc region of an immunoglobulin or indirectly through use of a short peptide linker, usually less than 20 amino acids in length, e.g., five amino acids, e.g., GGGGS, or 10 amino acids, e.g., GGGGSGGGGS.
  • nucleic acids that encode an AAT conjugated or fused to an Fc region of an immunoglobulin. As shown in FIG. 1, AAT can be conjugated to an Fc region of an IgG, in which case two AAT polypeptides (or therapeutically active variants thereof) are included in a single molecule.
  • AAT is synthesized in the liver and secreted into the plasma, where it is abundant at levels of approximately 1.5 to 3.5 mg/ml of blood.
  • the human AAT sequence is known in the art; exemplary reference sequences can be found in the GenBank database at accession number NM 000295.4 (nucleic acid) and NP 000286.3 (amino acid). See also GenelD: 5265.
  • AAT is encoded by a 1157 base pair sequence found on
  • chromosome 14 of the human genome (SEQ ID NO: l).
  • the pro-peptide as shown below, is 418 residues long (SEQ ID NO:2).
  • AAT is a single glycoprotein consisting of 394 amino acids with a molecular weight of 46,737 and an isoelectric point of 5.37. Further analysis reveals three potential sites to be
  • N-glycosylated and these three asparagine residues (N) are located at N70, N107, and N271.
  • the invention features nucleic acids encoding AAT-Fc fusion polypeptides, fragments, and variants thereof.
  • a nucleic acid sequence encoding an exemplary AAT is provided as SEQ ID NO: 1.
  • the amino acid sequence encoded by SEQ ID NO: 1 is provided as SEQ ID NO:2.
  • a nucleic acid sequence encoding an exemplary Fc region of an IgGl is provided as SEQ ID NO:3.
  • the amino acid sequence encoded by SEQ ID NO:3 is provided as SEQ ID NO:4.
  • the Fc region of an immunoglobulin is mutated to render it non- lytic.
  • four residues were mutated in the Fc region of a human IgGl : Leu-15-Glu; Glu-98-Ala; Cys-lOl-Ala; and Lys-102-Ala, as shown below as SEQ ID NO:6, and the non-lytic human IgGl is conjugated to an AAT polypeptide.
  • an AAT polypeptide is conjugated to an Fc region of IgGl at the N-terminus of AAT with two GGGGS linkers ("Construct 1") (FIG. 2A).
  • a nucleic acid sequence encoding an AAT polypeptide conjugated to an Fc region of IgGl at the N-terminus of AAT with two GGGGS linkers is provided as SEQ ID NO:7.
  • the amino acid sequence encoded by SEQ ID NO: 7 is provided as SEQ ID NO: 8, which comprises an IL2 signal peptide sequence, the sequence of an Fc region of IgGl, two GGGGS linkers, and the sequence of an AAT polypeptide.
  • the AAT polypeptide is directly conjugated to an Fc region of IgGl at the C-terminus of AAT ("Construct 2") (FIG. 2B).
  • a nucleic acid sequence encoding an AAT polypeptide directly conjugated to an Fc region of IgGl at the C terminus of AAT is provided as SEQ ID NO:9.
  • the amino acid sequence encoded by SEQ ID NO:9 is provided as SEQ ID NO:10, which comprises the sequence of an AAT polypeptide, an IgGl hinge sequence, and the sequence of an Fc region of IgGl .
  • the AAT polypeptide is conjugated to an Fc region of IgGl at the C-terminus of AAT with one GGGGS linker ("Construct 3") (FIG. 2C).
  • a nucleic acid sequence encoding an exemplary AAT polypeptide conjugated to an Fc region of IgGl at the C-terminus of AAT with one GGGGS linker is provided as SEQ ID NO: 11.
  • the amino acid sequence encoded by SEQ ID NO: 11 is provided as SEQ ID NO: 12, which comprises the sequence of an AAT polypeptide, a GGGGS linker, an IgGl hinge sequence, and the sequence of an Fc region of IgGl .
  • an AAT polypeptide is conjugated to a non-lytic Fc region of IgGl at the N-terminus of AAT with two GGGGS linkers ("Construct 4") (FIG.2A).
  • a nucleic acid sequence encoding an AAT polypeptide conjugated to a non-lytic Fc region of IgGl at the N-terminus of AAT with two GGGGS linkers is provided as SEQ ID NO: 13.
  • the amino acid sequence encoded by SEQ ID NO: 13 is provided as SEQ ID NO: 14, which comprises an IL2 signal peptide sequence, the sequence of a non-lytic Fc region of IgGl, two GGGGS linkers, and the sequence of an AAT polypeptide.
  • the AAT polypeptide is directly conjugated to a non-lytic Fc region of IgGl at the C-terminus of AAT ("Construct 5") (FIG. 2B).
  • a nucleic acid sequence encoding an AAT polypeptide directly conjugated to a non-lytic Fc region of IgGl at the C terminus of AAT is provided as SEQ ID NO: 15.
  • the amino acid sequence encoded by SEQ ID NO: 15 is provided as SEQ ID NO: 16, which comprises the sequence of an AAT polypeptide, an IgGl hinge sequence, and the sequence of a non-lytic Fc region of IgGl .
  • the AAT polypeptide is conjugated to a non-lytic Fc region of IgGl at the C-terminus of AAT with one GGGGS linker ("Construct 6") (FIG. 2C).
  • a nucleic acid sequence encoding an exemplary AAT polypeptide conjugated to a non-lytic Fc region of IgGl at the C-terminus of AAT with one GGGGS linker is provided as SEQ ID NO: 17.
  • the amino acid sequence encoded by SEQ ID NO: 17 is provided as SEQ ID NO: 18, which comprises the sequence of an AAT polypeptide, a GGGGS linker, an IgGl hinge sequence, and the sequence of a non-lytic Fc region of IgGl .
  • AAT-Fc nucleic acids described herein include both DNA and RNA, including genomic DNA and synthetic (e.g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can be synthesized using
  • oligonucleotide analogs or derivatives e.g., inosine or phosphorothioate nucleotides.
  • Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • isolated nucleic acid means a nucleic acid, e.g., DNA or RNA, that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated AAT-Fc nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to the AAT nucleic acid coding sequence.
  • the term includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.
  • the invention includes vectors, preferably expression vectors, containing a nucleic acid that encodes the fusion proteins described herein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include, e.g., a plasmid, cosmid, or viral vector.
  • the vector can autonomously replicate or it can integrate into a host cell's DNA.
  • Viral vectors include, e.g., replication-defective retroviruses, adenoviruses, and adeno- associated viruses.
  • a vector can include an AAT-Fc nucleic acid in a form suitable for expression of the nucleic acid in a host cell (FIG 3).
  • a recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
  • the term "regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce AAT-Fc polypeptides encoded by nucleic acids as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of AAT-Fc polypeptides in prokaryotic or eukaryotic cells.
  • polypeptides of the invention can be expressed in E. coli, insect cells ⁇ e.g., using baculovirus expression vectors), yeast cells, or mammalian cells ⁇ e.g., CHO or COS cells). Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, Gene 67:31-40, 1988), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • nucleic acid sequence of the nucleic acid is altered by standard DNA synthesis techniques.
  • peptide derivatives Modified versions of peptides disclosed herein are referred to as "peptide derivatives," and they can also be used in the new methods. For example, peptide derivatives of a peptide can be used instead of that peptide in therapeutic methods described herein. Peptides disclosed herein can be modified according to the methods known in the art for producing peptidomimetics. See, e.g., Kazmierski, W.M., ed.,
  • these modified peptidomimetic versions of the peptides and fragments disclosed herein exhibit enhanced stability in vivo, relative to the non- peptidomimetic peptides.
  • Methods for creating a peptidomimetic include substituting one or more, e.g., all, of the amino acids in a peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as "retro" sequences.
  • the N-terminal to C-terminal order of the amino acid residues is reversed, such that the order of amino acid residues from the N-terminus to the C-terminus of the original peptide becomes the order of amino acid residues from the C-terminus to the N-terminus in the modified peptidomimetic.
  • Such sequences can be referred to as "inverso" sequences.
  • Peptidomimetics can be both the retro and inverso versions, i.e., the
  • the new peptidomimetics can be composed of D-amino acids arranged so that the order of amino acid residues from the N-terminus to the C-terminus in the peptidomimetic corresponds to the order of amino acid residues from the C-terminus to the N-terminus in the original peptide.
  • peptidomimetics include replacing one or more amino acid residues in a peptide with a chemically distinct but recognized functional analog of the amino acid, i.e., an artificial amino acid analog.
  • Artificial amino acid analogs include ⁇ -amino acids, ⁇ -substituted ⁇ -amino acids (" 3 -amino acids”), phosphorous analogs of amino acids, such as a-amino phosphonic acids and a-amino phosphinic acids, and amino acids having non-peptide linkages.
  • Artificial amino acids can be used to create peptidomimetics, such as peptoid oligomers ⁇ e.g., peptoid amide or ester analogues), ⁇ -peptides, cyclic peptides, oligourea or oligocarbamate peptides; or heterocyclic ring molecules.
  • purified refers to a nucleic acid or polypeptide ⁇ e.g., an AAT-Fc nucleic acid or AAT-Fc polypeptide) that is substantially free of cellular or viral material with which it is naturally associated, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an isolated nucleic acid fragment is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
  • the invention includes nucleic acid sequences that are substantially identical to an AAT nucleic acid and to a nucleic acid encoding an Fc region of an immunoglobulin, e.g., an Fc region of an IgGl .
  • a nucleic acid sequence that is "substantially identical" to an AAT nucleic acid is at least 90% identical ⁇ e.g., at least or about 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the AAT nucleic acid sequence represented by SEQ ID NO: 1.
  • a nucleic acid sequence that is "substantially identical" to a nucleic acid encoding an Fc region of an IgGl is at least 90% identical ⁇ e.g., at least or about 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence represented by SEQ ID NO: 3 or 5.
  • the length of the reference nucleic acid sequence will be at least 50 nucleotides, but can be longer, e.g., at least 60 or more nucleotides.
  • the sequences are aligned for optimal comparison purposes ⁇ i.e., gaps can be introduced as required in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences may be of the same length.
  • the percent identity or homology between two sequences can be determined using a mathematical algorithm.
  • a non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc Natl Acad Sci USA 87:2264-2268, 1990, modified as in Karlin and Altschul, Proc Natl Acad Sci USA 90:5873-5877, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, J Mol Biol 215:403-410, 1990.
  • BLAST protein searches can be performed with the
  • Gapped BLAST can be utilized as described in Altschul et al. , Nucleic Acids Res. 25:3389-3402, 1997.
  • the default parameters of the respective programs ⁇ e.g., XBLAST and
  • NBLAST NBLAST
  • the invention includes variants, homologs, and/or fragments of certain AAT nucleic acids, e.g., variants, homologs, and/or fragments of the AAT nucleic acid sequences represented by SEQ ID NO: 1.
  • the terms "variant” or “homolog” in relation to AAT nucleic acids include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleotides from or to the sequence of an AAT nucleic acid.
  • the resultant nucleotide sequence may encode an AAT polypeptide that has at least 50% of a biological activity ⁇ e.g., inhibition of elastase) of the referenced AAT polypeptides ⁇ e.g., SEQ ID NO:2).
  • homolog covers homology with respect to structure and/or function as long as the resultant nucleotide sequence encodes or is capable of encoding an AAT polypeptide that has at least 50% of the biological activity of AAT encoded by a sequence shown herein as SEQ ID NO: 1. With respect to sequence homology, there is at least 75% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) homology to the sequence shown as SEQ ID NO: 1.
  • sequence homology there is at least 75% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) homology to the sequence shown as SEQ ID NO: 1.
  • the term “homology” as used herein can be equated with the term "identity.”
  • “Substantial homology” or “substantially homologous,” where homology indicates sequence identity, means at least 90%> identical (e.g., at least about 92%, 95%, 96%), 97%), 98%), or 99%) sequence identity, as judged by direct sequence alignment and comparison.
  • “Substantial homology” when assessed by the BLAST algorithm equates to sequences which match with an EXPECT value of at least about 7, e.g., at least about 9, 10, or more.
  • the default threshold for EXPECT in BLAST searching is usually 10.
  • an "allele” or “allelic sequence” is an alternative form of AAT. Alleles can result from changes in the nucleotide sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene can have none, one, or more than one allelic form. Common changes that give rise to alleles are generally ascribed to deletions, additions, or substitutions of amino acids. Each of these types of changes can occur alone, or in combination with the others, one or more times in a given sequence.
  • the invention also includes nucleic acids that hybridize, e.g., under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequences represented by SEQ ID NO: 1 , or a complement thereof.
  • the hybridizing portion of the hybridizing nucleic acids is typically at least 15 (e.g., 20, 30, or 50) nucleotides in length.
  • the hybridizing portion of the hybridizing nucleic acid is at least about 75%, e.g., at least about 80%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of a portion or all of a nucleic acid encoding an AAT polypeptide, or to its complement.
  • Hybridizing nucleic acids of the type described herein can be used as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe.
  • Nucleic acids that hybridize to the nucleotide sequence represented by SEQ ID NO: l are considered "antisense
  • oligonucleotides . ' ' High stringency conditions are hybridizing at 68°C in 5x SSC/5x Denhardt's solution/1.0% SDS, or in 0.5 M NaHP0 4 ( H ⁇ 2) ⁇ niM EDTA/7% SDS, or in 50% formamide/0.25 M NaHP0 4 (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; and washing in 0.2x SSC/0.1% SDS at room temperature or at 42 °C, or in O.
  • genetic constructs ⁇ e.g., vectors and plasmids
  • an AAT-Fc nucleic acid described herein operably linked to a transcription and/or translation sequence to enable expression, e.g., expression vectors.
  • a selected nucleic acid e.g., a DNA molecule encoding an AAT-Fc polypeptide
  • a transformed cell is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding an AAT polypeptide.
  • AAT-Fc nucleic acid encoding an AAT polypeptide.
  • prokaryotic and eukaryotic cells are included.
  • Mammalian cells transformed with an AAT-Fc nucleic acid can include host cells for an attaching enteric organism, e.g., intestinal cells, HeLa cells, and mouse embryonic fibroblasts.
  • Prokaryotic cells can include bacteria, e.g.,
  • Escherichia coli An engineered cell exemplary of the type included in the invention is an E. coli strain that expresses AAT.
  • Certain chimeric AAT polypeptides are included within the present invention.
  • Examples of such chimeric AAT polypeptides are AAT polypeptides and fragments, such as the one shown as SEQ ID NO:2 conjugated to an Fc region of an immunoglobulin, e.g., IgGl, shown as SEQ ID NO:4 and 6.
  • Also included within the present invention are certain fragments of AAT polypeptides, e.g., fragments of AAT polypeptides may inhibit elastase, or other useful portions of a full-length AAT polypeptide.
  • useful fragments of AAT polypeptides include, but are not limited to, fragments having elastase-inhibiting activity, and portions of such fragments.
  • protein and “polypeptide” both refer to any chain of amino acids, regardless of length or post-translational modification ⁇ e.g., glycosylation or
  • AAT includes full-length naturally occurring isolated proteins, as well as recombinantly or synthetically produced polypeptides that correspond to the full-length naturally occurring proteins, or to a fragment of the full-length naturally occurring or synthetic polypeptide. Fragments of a protein can be produced by any of a variety of methods known to those skilled in the art, e.g., recombinantly, by proteolytic digestion, and/or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid that encodes the polypeptide.
  • DNAs that encode fragments of a protein can also be generated, e.g., by random shearing, restriction digestion, chemical synthesis of oligonucleotides, amplification of DNA using the polymerase chain reaction, or a combination of the above-discussed methods. Fragments can also be chemically synthesized using techniques known in the art, e.g., conventional Merrifield solid phase FMOC or t-Boc chemistry. For example, peptides of the present invention can be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
  • a purified or isolated compound is a composition that is at least 75% by weight the compound of interest, e.g., AAT.
  • the preparation is at least 80% ⁇ e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) by weight the compound of interest.
  • Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • AAT includes sequences substantially identical to all or portions of a naturally occurring AAT polypeptide.
  • Polypeptides "substantially identical" to the AAT polypeptide sequences described herein have an amino acid sequence that is at least 75% (e.g., at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)) identical to the amino acid sequences of the AAT polypeptides represented by SEQ ID NO:2 (measured as described herein).
  • the length of the reference AAT polypeptide sequence is at least 50 amino acids, e.g., at least 60, 80, 100, 200, 300, 394, or 418 amino acids.
  • non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence.
  • conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a polypeptide that has 50%> identity to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It also might be a 100 amino acid long polypeptide that has 50%> identity to the reference polypeptide over its entire length.
  • the Fc region can be mutated to inhibit its ability to fix complement and bind the Fc receptor.
  • substitution of Ala residues for Glu 318, Lys 320, and Lys 322 renders the protein unable to direct ADCC.
  • the "Fc region” can be a naturally-occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by digestion of IgG with papain.
  • the polypeptide agents described herein can include the entire Fc region or a smaller portion that retains the ability to extend the circulating half-life of a chimeric polypeptide of which it is a part.
  • full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptide; as described further below, native activity is not necessary or desired in all cases.
  • the Fc region includes the hinge, CH2 and CH3 domains of human IgGl or murine IgG2a.
  • the Fc region can be isolated from a naturally occurring source, recombinantly produced, or synthesized (as any polypeptide featured in the present invention can be).
  • an Fc region that is homologous to the IgG C-terminal domain can be produced by digestion of IgG with papain.
  • the polypeptides of the invention can include the entire Fc region, or a smaller portion that retains the ability to lyse cells.
  • full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptide.
  • Chimeric AAT-Fc polypeptides can be constructed using no more than conventional molecular biological techniques, which are well within the ability of those of ordinary skill in the art to perform.
  • protein and
  • polypeptide both refer to any chain of amino acid residues, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation).
  • AAT-Fc polypeptide one can, for example, subclone cDNA of AAT and an Fc region of an immunoglobulin into the pFUFC vector from InvivoGen to create an AAT-Fc fusion protein; the AAT-Fc portion of this plasmid can be cloned into the UCOE expression vector from Millipore; and stable human AAT expressed in CHO cells can be screened by ELISA.
  • Large amounts of AAT fusion protein can be produced by the WAVE bioreactor system, and purification can be achieved with protein A affinity chromatography columns.
  • the UCOE (ubiquitous chromatin opening element) expression system can be employed because it gives major improvements in gene expression in stably-transfected mammalian cells.
  • AAT activity can be measured in terms of the inhibitory capacity of AAT toward trypsin.
  • Microplates can be coated with 1% of FBS and incubated for one hour. After a wash (3X) with H2O, various concentrations of AAT can be incubated with a fixed amount of trypsin for 20 minutes at 37°C in a 200 ⁇ final reaction volume.
  • chromogenic substrate L-pyroglutamylglycyl-L-arginine, p-nitroanilide hydrochloride
  • chromogenic substrate L-pyroglutamylglycyl-L-arginine, p-nitroanilide hydrochloride
  • the reaction can be stopped with 50 ⁇ of 50% acetic acid.
  • Absorbance can be read at 400 nm in a microplate reader.
  • Nucleic Acid Molecules That Encode Agents of the Invention Polypeptide agents of the invention, including those that are fusion proteins ⁇ e.g., AAT-Fc, as discussed herein) cannot only be obtained by expression of a nucleic acid molecule in a suitable eukaryotic or prokaryotic expression system in vitro and subsequent purification of the polypeptide agent, but can also be administered to a patient by way of a suitable gene therapeutic expression vector encoding a nucleic acid molecule. Further, a nucleic acid can be introduced into a cell of a graft prior to transplantation of the graft. Thus, nucleic acid molecules encoding the agents described above are within the scope of the invention.
  • nucleic acid molecules encoding them will necessarily have a certain identity with those that encode the corresponding wild-type polypeptides.
  • the nucleic acid molecule encoding a cytokine polypeptide is at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 90% ⁇ e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleic acid encoding wild-type cytokine.
  • the length of the sequences compared will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
  • nucleic acid molecules that encode agents of the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide.
  • These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids.
  • the nucleic acid molecules can be double-stranded or single-stranded (i.e., either a sense or an antisense strand).
  • nucleic acid molecules of the invention may be referred to as "isolated" when they are separated from either the 5 ' or the 3 ' coding sequence with which they are immediately contiguous in the naturally occurring genome of an organism.
  • the nucleic acid molecules are not limited to sequences that encode polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence can also be included.
  • Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by
  • nucleic acid molecule is a RNA
  • molecules can be produced by in vitro transcription.
  • the isolated nucleic acid molecules of the invention can include fragments not found as such in the natural state.
  • the invention encompasses recombinant molecules, such as those in which a nucleic acid sequence is incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).
  • agents of the invention can be fusion proteins.
  • a nucleic acid molecule encoding an agent of the invention can contain sequences encoding a "marker” or "reporter.”
  • marker or reporter genes include ⁇ -lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
  • phosphotransferase (neo r , G418 r ), dihydro folate reductase (DHFR), hygromycin-B- phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding ⁇ -galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).
  • DHFR dihydro folate reductase
  • HPH hygromycin-B- phosphotransferase
  • TK thymidine kinase
  • lacZ encoding ⁇ -galactosidase
  • XGPRT xanthine guanine phosphoribosyltransferase
  • nucleic acid molecules described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Accordingly, in addition to polypeptide agents, expression vectors containing a nucleic acid molecule encoding those agents and cells transfected with those vectors are among the preferred embodiments.
  • Vectors suitable for use in the present invention include T7-based vectors for use in bacteria (see, e.g., Rosenberg et al., Gene 56: 125, 1987), the pMSXND expression vector for use in mammalian cells (Lee and Nathans, J Biol Chem 263:3521, 1988), yeast expression systems, such as Pichia pastoris (for example the PICZ family of expression vectors from Invitrogen, Carlsbad, CA) and baculovirus-derived vectors (for example the expression vector pBacPAK9 from Clontech, Palo Alto, CA) for use in insect cells.
  • yeast expression systems such as Pichia pastoris (for example the PICZ family of expression vectors from Invitrogen, Carlsbad, CA) and baculovirus-derived vectors (for example the expression vector pBacPAK9 from Clontech, Palo Alto, CA) for use in insect cells.
  • the nucleic acid inserts which encode the polypeptide of interest in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.
  • a promoter which is selected based on, for example, the cell type in which expression is sought.
  • a T7 promoter can be used in bacteria
  • a polyhedrin promoter can be used in insect cells
  • metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body.
  • tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body.
  • tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body.
  • One of ordinary skill in the art is well aware of numerous promoters and other regulatory elements that can be used to direct expression of nucleic acids.
  • vectors can contain origins of replication, and other genes that encode a selectable marker.
  • neomycin-resistance (neo 1 ) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells.
  • Other feasible selectable marker genes allowing for phenotypic selection of cells include various fluorescent proteins, e.g. green fluorescent protein (GFP) and variants thereof.
  • GFP green fluorescent protein
  • Viral vectors that can be used in the invention include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, New York).
  • Prokaryotic or eukaryotic cells that contain a nucleic acid molecule that encodes an agent of the invention and express the protein encoded in that nucleic acid molecule in vitro are also features of the invention.
  • a cell of the invention is a transfected cell, i.e., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a polypeptide, has been introduced by means of recombinant DNA techniques.
  • the progeny of such a cell are also considered within the scope of the invention.
  • the precise components of the expression system are not critical.
  • a polypeptide can be produced in a prokaryotic host, such as the bacterium E.
  • coli or in a eukaryotic host, such as an insect cell (for example, Sf21 cells), or mammalian cells (e.g., COS cells, CHO cells, 293 cells, NIH 3T3 cells, or HeLa cells).
  • insect cell for example, Sf21 cells
  • mammalian cells e.g., COS cells, CHO cells, 293 cells, NIH 3T3 cells, or HeLa cells.
  • Eukaryotic cells that contain a nucleic acid molecule that encodes the agent of the invention and express the protein encoded in such nucleic acid molecule in vivo are also features of the invention.
  • eukaryotic cells of the invention can be cells that are part of a cellular transplant, a tissue or organ transplant.
  • Such transplants can comprise either primary cells taken from a donor organism or cells that were cultured, modified and/or selected in vitro before transplantation to a recipient organism (e.g., eukaryotic cells lines, including stem cells or progenitor cells). Since, after transplantation into a recipient organism, cellular proliferation may occur, the progeny of such a cell are also considered within the scope of the invention.
  • a cell, being part of a cellular, tissue or organ transplant can be transfected with a nucleic acid encoding a polypeptide of interest and subsequently be transplanted into the recipient organism, where expression of the polypeptide occurs.
  • such a cell can contain one or more additional nucleic acid constructs allowing for application of selection procedures, e.g. of specific cell lineages or cell types prior to transplantation into a recipient organism.
  • the expressed polypeptides can be purified from the expression system using routine biochemical procedures, and can be used as diagnostic tools or as therapeutic agents, as described below.
  • growth media and purification and isolation reagents were supplemented with methionine and used to produce and isolate the proteins.
  • methionine-rich proteins e.g., AAT, AAT conjugated to an Fc region, or AAT conjugated to other protein(s)
  • special growth media i.e., growth media that include high levels of methionine, e.g., a final concentration of at least 10 mM, e.g., at least 20 mM, 50 mM, 100 mM, 200 mM, or 500 mM, methionine.
  • special purification or isolation reagents e.g., extraction buffers, wash buffers, and elution buffers
  • the methods include providing a cell comprising a nucleic acid molecule encoding a polypeptide comprising AAT or AAT conjugated to an Fc region of an immunoglobulin or conjugated to other protein(s); and culturing the cell under conditions sufficient to produce AAT or AAT conjugated to an Fc region of an immunoglobulin or conjugated to other protein(s), e.g., in growth media having a concentration of at least 10 mM, e.g., at least 20 mM, 50 mM, 100 mM, 200 mM, or 500 mM, methionine, thereby producing AAT or AAT conjugated to an Fc region of an immunoglobulin or conjugated to other protein(s).
  • the method may include purifying or isolating from the cell AAT or AAT conjugated to an Fc region or other protein(s) using at least one purification and/or isolation reagent having a concentration of at least 10 mM, e.g., at least 20 mM, 50 mM, 100 mM, 200 mM, or 500 mM, methionine. It will be appreciated that methods of the present invention can include growth of the cells in the methionine -rich medium and isolation using standard purification and/or isolation reagents.
  • the methods can include growth of the cell in standard growth medium, followed by isolation using the special purification or isolation reagent(s), e.g., extraction buffers, wash buffers, and elution buffers, that are supplemented with methionine.
  • methods can be carried out using growth in methionine-rich medium followed by isolation using one or more special purification or isolation reagents that are supplemented with methionine.
  • Human AAT-Fc plasmids can be transfected into the CHO-S Chinese hamster ovary cell line.
  • AAT-Fc fusion proteins were expressed in a CHO cell line transfected with AAT-Fc plasmids.
  • Transfected CHO cells were cultured in CD CHO medium (Gibco), a protein-free, serum-free medium, supplemented with 50 mM L-methionine. The proteins and purified by standard protein A chromatographic procedures using loading (pH 7) and elution (pH 2.1 to 3.2) conditions and the addition on 50 mM
  • Stable clones are selected and subsequently subjected to selection for gene amplification.
  • Stable clones that secrete soluble recombinant AAT-Fc can be grown in serum-free medium.
  • Single cell clones can be selected and cultured.
  • the culture medium is first filtered to remove debris.
  • the filtrate can then be purified with protein A-conjugated beads that have been equilibrated with phosphate-buffered saline (PBS) containing 0.5 mol/L NaCl.
  • PBS phosphate-buffered saline
  • the filtered cell culture medium can be passed through a column containing the protein A resin, and 1 mL fractions of purified protein collected.
  • Glycine pH 2.7
  • aliquots of purified protein fractions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels.
  • Resolved proteins can be transferred to a nitrocellulose membrane and Western blotting analysis performed using rabbit anti-human AAT (YbdY, Seoul Korea) and horse radish peroxidase-conjugated goat anti-human IgG Fc antibodies (Sigma-Aldrich, St. Louis, MO, USA).
  • Protein-antibody complexes can be detected using Supex (Neuronex, Seoul, Korea) and an LAS-4000 imaging device (Fujifilm, Japan).
  • the SENSOLYTE ® Green Elastase Assay Kit (Anaspec, Inc., Fremont, CA) is a FRET-based assay that detects elastase activity.
  • the kit provides an elastin, a natural substrate for elastases, labeled with 5-FAM fluorophore and QXL TM 520 quencher.
  • Elastase-catalyzed hydrolysis yields brightly green fluorescence.
  • An increase in fluorescence intensity is directly proportional to enzyme activity. Diminution of fluorescence intensity in the presence of AAT indicates elastase inhibition via AAT action.
  • Activity was analyzed by a SPECTRAMAX M5 ® instrument (Molecular Devices, Sunnyvale, CA).
  • a no-acid condition refers to a commercial formulation of AAT used in the assay without any exposure to acidic pH.
  • acidic conditions either a commercial formulation of AAT exposed to acidic conditions (e.g., pH 2.1) or an AAT-Fc fusion protein purified under acidic conditions will be assayed for elastase inhibition assay.
  • Elastase is supplied in a commercial kit.
  • compositions which include AAT conjugated to an Fc region of an immunoglobulin, e.g., where the AAT comprises a polypeptide that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2 and the Fc region of the immunoglobulin comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4 or 6, and vectors encoding an AAT conjugated to an Fc region of an immunoglobulin, e.g., wherein the nucleic acid sequence encoding AAT is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 and the nucleic acid sequence encoding the Fc region of the immunoglobulin is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:
  • a pharmaceutical composition can comprise a polypeptide comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8, 10, 12, 14, 16, or 18, and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprise a vector comprising a nucleic acid molecule encoding a recombinant polypeptide comprising an AAT comprising an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 conjugated to an Fc region of an immunoglobulin and a
  • the nucleic acid molecule encoding AAT comprises a nucleic acid sequence that is at least 90%>, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: l .
  • the nucleic acid molecule comprises an AAT-Fc nucleic acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:7, 9, 11, 13, 15, or 17.
  • compositions typically include the active compound and a pharmaceutically acceptable carrier.
  • a "pharmaceutically acceptable carrier” can include one or more solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediammetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or a suitable mixture thereof.
  • a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or a suitable mixture thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be achieved by including an agent that delays absorption, e.g., aluminum monostearate or gelatin, in the composition.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Injectable compositions may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • the compounds may be administered by the drip method, whereby a pharmaceutical composition containing the active compound(s) and a physiologically acceptable excipient is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
  • a sterile composition of a suitable soluble salt form of the compound can be dissolved and administered in a pharmaceutical excipient such as Water- for-Injection, 0.9% saline, or 5% glucose solution, or depot forms of the compounds (e.g., decanoate, palmitate, undecylenic, enanthate) can be dissolved in sesame oil.
  • a pharmaceutical excipient such as Water- for-Injection, 0.9% saline, or 5% glucose solution
  • depot forms of the compounds e.g., decanoate, palmitate, undecylenic, enanthate
  • Oral compositions typically include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • the pharmaceutical composition can be formulated as a chewing gum, lollipop, or the like.
  • Liquid compositions for oral administration prepared in water or other aqueous vehicles can include solutions, emulsions, syrups, and elixirs containing, together with the active compound(s), wetting agents, sweeteners, coloring agents, and flavoring agents.
  • Various liquid and powder compositions can be prepared by conventional methods for inhalation into the lungs of the patient to be treated.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as known in the art.
  • the compounds can also be prepared in the form of suppositories ⁇ e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories ⁇ e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • compositions of the invention are useful in inhibiting T cells that are involved, or would be involved, in an immune response ⁇ e.g., a cellular immune response) to an antigen; in inhibiting other cells involved in the pathogenesis of immunological disorders ⁇ e.g., monocytes, macrophages, and other antigen presenting cells such as dendritic cells, NK cells, and granulocytes); and in destroying cells such as islet cells (as seen in diabetes), or hyperproliferating cells (as seen, for example, in tissues involved in immunological disorders such as synovial fibroblasts (which are affected in rheumatoid arthritis) keratinocytes (which are affected in psoriasis), or dermal fibroblasts (which are affected in systemic lupus erythematosus).
  • an immune response ⁇ e.g., a cellular immune response
  • an antigen e.g., monocytes, macrophages, and other antigen presenting cells
  • compositions of the invention can be used to treat patients who are suffering from, or at risk for, an immune disease, particularly autoimmune disease.
  • autoimmune diseases suitable for treatment are Type 1 or Type 2 diabetes, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue- dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic
  • inflammatory demyelinating polyneuropathy Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), irritable bowel disease (IBD), IgA
  • neuropathy juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma,
  • Sjogren's syndrome stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/ giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
  • Inflammatory conditions which are often, but not always, associated with autoimmunity
  • Inflammatory conditions which may be amenable to treatment are asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic inflammation resulting from chronic viral or bacterial infections, psoriasis (e.g., plaque psoriasis, pustular psoriasis, erythrodermic psoriasis, guttate psoriasis or inverse psoriasis).
  • COPD chronic obstructive pulmonary disease
  • transplants can be organ, tissue or cell transplants, or synthetic grafts seeded with cells, for example, synthetic vascular grafts seeded with vascular cells.
  • synthetic vascular grafts seeded with vascular cells.
  • patients suffering from GVHD or patients who have received a vascular injury would benefit from this method.
  • compositions can be used to treat patients at risk for, or diagnosed with, Type 1 diabetes.
  • compositions are also useful for treating patients at risk for, or suffering from, Type 2 diabetes.
  • the invention encompasses administration of target-cell depleting forms of an agent that targets tissue destructive T cells, or inflammatory cells.
  • target-cell depleting forms of agents it is possible to selectively kill autoreactive or "transplant destructive" immune cells without massive destruction of other subsets of T cells (e.g., regulatory T cells).
  • the invention features a method of killing cells (e.g., autoreactive Thl7 cells, or proinflammatory effector cells such as macrophages). These methods can be carried out by administering to a patient a combination of agents that includes an agent that activates the complement system, lyses cells by the ADCC mechanism, or otherwise kills cells expressing a selected target molecule.
  • compositions and methods are clearly contemplated for use in human patients, the invention is not so limited.
  • the compositions and methods can be used in veterinary settings as well (e.g. , to treat a domesticated animal such as a dog, cat, or horse).
  • agents of the present invention can be obtained from naturally occurring sources, they can also be synthesized or otherwise manufactured. Polypeptides that are derived from eukaryotic organisms or synthesized in E. coli, or other prokaryotes, and polypeptides that are chemically synthesized will be substantially free from their naturally associated components. In the event the polypeptide is a chimera, it can be encoded by a hybrid nucleic acid molecule containing one sequence that encodes all or part of the agent.
  • Agents of the invention can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
  • codons can be optimized based on the codon preference of the host cell.
  • agents of the invention can be administered with a physiologically acceptable carrier, such as physiological saline.
  • the therapeutic compositions of the invention can also contain a carrier or excipient, many of which are known to one of ordinary skill in the art. Excipients that can be used include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol.
  • the agents of the invention can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for ingestion or injection; gels or powders can be made for ingestion, inhalation, or topical application. Methods for making such formulations are well known and can be found in, for example,
  • Routes of administration are also well known to skilled pharmacologists and physicians and include intraperitoneal, intramuscular, subcutaneous, and intravenous administration. Additional routes include intracranial (e.g., intracisternal or
  • intraventricular intraventricular
  • intraorbital intraorbital
  • opthalmic intracapsular
  • intraspinal intraperitoneal
  • transmucosal topical, subcutaneous, and oral administration.
  • the subcutaneous route may also be used frequently as the subcutaneous tissue provides a stable environment for polypeptides, from which they can be slowly released.
  • the cells/tissues/organs could either be transfected by incubation, infusion or perfusion prior to transplantation with a nucleic acid composition, such that the therapeutic protein is expressed and subsequently released by the transplanted cells/tissues/organs within the recipient organism.
  • the cells/tissues/organs could undergo a pretreatment by perfusion or simple incubation with the therapeutic protein prior to transplantation in order to eliminate transplant- associated immune cells adherent to the donor cells/tissues/organs.
  • the cells may be administered either by an implantation procedure or with a catheter-mediated injection procedure through the blood vessel wall. In some cases, the cells may be administered by release into the vasculature.
  • dosages for any one patient depend on many factors, including the general health, sex, weight, body surface area, and age of the patient, as well as the particular compound to be administered, the time and route of administration, and other drugs being administered concurrently.
  • Dosages for the polypeptide of the invention will vary, but can, when administered intravenously, be given in doses on the order of magnitude of 1 microgram to lOmg/kg body weight or on the order of magnitude of O.Olmg/1 to l OOmg/1 of blood volume.
  • a dosage can be administered one or more times per day, if necessary, and treatment can be continued for prolonged periods of time. Determining the correct dosage for a given application is well within the abilities of one of ordinary skill in the art.
  • appropriate dosages of AAT-Fc can readily be determined by those of ordinary skill in the art of medicine, e.g., by monitoring the patient for signs of disease amelioration or inhibition, and increasing or decreasing the dosage and/or frequency of treatment as desired.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to thereby, reduce side effects.
  • an "effective amount” is an amount sufficient to effect beneficial or desired results.
  • a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a composition depends on the
  • the compositions can be administered from one or more times per day to one or more times per week; including once every other day.
  • the skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of the compositions described herein can include a single treatment or a series of treatments.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • an effective amount e.g., of a protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, e.g., about 0.01 to 25 mg/kg body weight, e.g., about 0.1 to 20 mg/kg body weight.
  • the protein or polypeptide can be administered one time per day, twice per day, one time per week, twice per week, for between about 1 to 52 weeks per year, e.g., between 2 to 50 weeks, about 6 to 40 weeks, or for about 4, 5, or 6 weeks.
  • treatment of a patient with a therapeutically effective amount of a protein, polypeptide, nucleic acid, or other compound can include a single treatment or, preferably, can include a series of treatments.
  • the AAT-Fc polypeptide described herein is to be administered to an animal (e.g., a human), a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • an animal e.g., a human
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • Nucleic acid molecules (e.g., encoding an AAT-Fc fusion polypeptide) of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al, Proc Natl Acad Sci USA 91 :3054-3057, 1994).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • subjects amenable to treatment include those that exhibit pancreatic ⁇ cell loss or functional insufficiency. These subjects, particularly when they also exhibit impaired glucose tolerance, are at risk of developing Type 1 diabetes or are at the inflection point between a diabetic and non-diabetic state.
  • One of ordinary skill in the art will recognize that there is a progression of events associated with oncoming illness and that patients can be treated at varying points along the continuum.
  • the compositions and methods described herein can also be used to treat subjects who are insulin resistant.
  • Insulin resistant subjects include those who have Type 2 diabetes (a condition understood to be associated with insulin resistance), subjects who are at risk of developing Type 2 diabetes, and subjects diagnosed as having metabolic syndrome.
  • the subjects also include insulin deficient patients with Type 1 diabetes, marginal islet function, and insulin resistance.
  • Models of Autoimmune Disease provide another means to assess combinations of the agents of the invention in vivo. These models are well known to those of ordinary skill in the art and can be used to determine whether a given combination of agents would be therapeutically useful in treating a specific autoimmune disease when delivered either directly, via genetic therapy, or via cell-based therapies.
  • rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE), Type 1 diabetes, and autoimmune diseases of the thyroid, gut, and central nervous system.
  • SLE systemic lupus erythematosus
  • autoimmune diseases of the thyroid, gut, and central nervous system include rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE), Type 1 diabetes, and autoimmune diseases of the thyroid, gut, and central nervous system.
  • animal models of SLE include MRL mice, BXSB mice, and NZB mice and their Fi hybrids. These animals can be crossed in order to study particular aspects of the rheumatic disease process; progeny of the NZB strain develop severe lupus
  • mice with targeted deletions for IL-2, IL-10, MHC class II or TCR genes among others include mutant mice with targeted deletions for IL-2, IL-10, MHC class II or TCR genes among others.
  • MRL-lpr/lpr One of the MRL strains of mice that develops SLE, MRL-lpr/lpr, also develops a form of arthritis that resembles rheumatoid arthritis in humans (Theofilopoulos et al. , Adv Immunol 37:269, 1985).
  • an experimental arthritis can be induced in rodents by injecting rat type II collagen (2 mg/ml) mixed 1 : 1 in Freund's complete adjuvant (100 ⁇ total) into the base of the tail. Arthritis develops 2-3 weeks after immunization. The effectiveness of a candidate treatment is assessed by following the disease symptoms during the subsequent 2 weeks, as described by Chernajovsky et al. (Gene Therapy 2:731-735, 1995). Lesser symptoms, compared to control, indicate that the combined agents of the invention, and the nucleic acid molecules that encode them, function as immunosuppressants and are therefore useful in the treatment of immune disease, particularly autoimmune disease.
  • OS chickens consistently develop spontaneous autoimmune thyroiditis resembling Hashimoto's disease (Cole et al., Science 160: 1357, 1968). Approximately 15% of these birds produce autoantibodies to parietal cells of the stomach, just as in the human counterpart of autoimmune thyroiditis.
  • the manifestations of the disease in OS chickens include body size, fat deposit, serum lipids, cold sensitivity, and infertility.
  • Models of autoimmune disease in the central nervous system can also be experimentally induced.
  • An inflammation of the CNS which leads to paralysis, can be induced by a single injection of brain or spinal cord tissue with adjuvant in many different laboratory animals, including rodents and primates.
  • This model referred to as experimental allergic encephalomyelitis (EAE) is T cell mediated.
  • EAE experimental allergic encephalomyelitis
  • mice can also be produced by a single injection of acetylcholine receptor with adjuvants (Lennon et ⁇ , ⁇ . N. Y. Acad. Sci. 274:283, 1976).
  • Islet Allograft Model DBA/2J islet cell allografts can be transplanted into rodents, such as 6-8 week-old B6 AF1 mice rendered diabetic by a single intraperitoneal injection of streptozotocin (225 mg/kg; Sigma Chemical Co., St. Louis, MO). As a control, syngeneic islet cell grafts can be transplanted into diabetic mice. Islet cell transplantation can be performed by following published protocols (for example, see Gotoh et al., Transplantation 42:387, 1986). Briefly, donor pancreata are perfused in situ with type IV collagenase (2 mg/ml; Worthington Biochemical Corp., Freehold, NJ).
  • the islets are isolated on a discontinuous Ficoll gradient. Subsequently, 300-400 islets are transplanted under the renal capsule of each recipient. Allograft function can be followed by serial blood glucose measurements (ACCU-CHECK III TM ; Boehringer, Mannheim, Germany).
  • Primary graft function is defined as a blood glucose level under 11.1 mmol/1 on day 3 post-transplantation, and graft rejection is defined as a rise in blood glucose exceeding 16.5 mmol/1 (on each of at least two successive days) following a period of primary graft function.
  • Constructs 1 and 4 were cloned in pFuse-hIgG-Fc2, and Constructs 2, 3, 5, and 6 were cloned in pFuse-hlgG-Fcl .
  • Constructs 4 5, and 6, the wild type hlgGl-Fc sequence in pFuse vector backbone was replaced with a non- lytic hlgGl- Fc version.
  • the non-lytic IgGl-Fc (with mutant residues: Leu-15-Glu; Glu-98-Ala; Cys- 101-Ala; and Lys-102-Ala) was released from an earlier construct and cloned between EcoRV and Ncol in Construct 4 or between Bglll and Nhel in Constructs 5 and 6.
  • Construct 1 wild type hlgGl-Fc was cloned between EcoRV and Ncol.
  • the IgGl hinge and/or GGGGS linker sequences were cloned using annealed synthetic oligonucleotides between Ncol and Bglll (FIG. 4).
  • cDNA coding human AAT was amplified using cDNA clones available from Open Biosystems, using the following primers.
  • AAT either commercial AAT or AAT-Fc fusion protein
  • concentrations of AAT in the range of 1 to 200 nM were tested for the ability to inhibit elastase activity in a 96-well microtiter plate assay. Briefly, 40 ⁇ of AAT and 10 ⁇ of elastase solution were added to each well of a flat-bottom black 96-well microtiter plate, followed by the addition of 50 ⁇ of the fluorescent substrate for elastase. Separate wells were maintained for test protein /inhibitor control, and enzyme and substrate controls were included. The contents of the wells were mixed for 30 seconds and incubated at room temperature for 30-60 minutes. At the end of the incubation period, the plate was read at

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Abstract

La description concerne des compositions comprenant des protéines chimériques comprenant une AAT conjuguée à une région Fc d'une immunoglobuline. L'invention concerne également des méthodes de traitement d'une maladie auto-immune, par ex., le diabète, par ex., les diabètes de Type 1 et de Type 2.
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EP3914614A1 (fr) * 2019-01-24 2021-12-01 Excellgene SA Procédés de production et d'utilisation d'alpha 1-antitrypsine (aat) de recombinaison et compositions associées
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US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US5328470A (en) 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
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EP2069396B1 (fr) * 2006-09-08 2015-10-21 Ambrx, Inc. Polypeptide plasmatique humain modifie ou squelettes de fc et leurs utilisations
US20140147441A1 (en) * 2006-09-12 2014-05-29 The General Hospital Corporation Compositions containing alpha-1-antitrypsin and methods for use
WO2012087835A2 (fr) * 2010-12-22 2012-06-28 Boston Biomedical Research Institute Compositions et procédés pour favoriser le repliement d'une protéine
JP6216921B2 (ja) * 2011-06-24 2017-11-01 ザ リージェンツ オブ ザ ユニバーシティ オブ コロラド，ア ボディー コーポレイトＴＨＥ ＲＥＧＥＮＴＳ ＯＦ ＴＨＥ ＵＮＩＶＥＲＳＩＴＹ ＯＦ ＣＯＬＯＲＡＤＯ，ａ ｂｏｄｙ ｃｏｒｐｏｒａｔｅ アルファ−１抗トリプシン融合分子のための組成物、方法および使用
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